Among our observations during the past three years, we have shown that the Chklp two-component histidine kinase [HK] of Candida albicans regulates cell wall biosynthesis. These data were achieved through an analysis of the chkl deletion mutant. The consequences of the cell wall changes are profound as the chkl mutant is less able to adhere to human esophageal tissue in vitro, is more susceptible to growth inhibition and killing by human neutrophils and hydrogen peroxide, and, importantly, is avirulent. Also, the chkl mutant is refractory to the C. albicans quorum sensing molecule, farnesol. In addition to the Chklp, C. albicans has two other HK proteins, Slnlp and Nik1/Cos1p, and our studies have shown that each of the HKs regulate common functions but also have specific functions. For example, all HKs apparently regulate mannosylation of the cell wall acid-stable mannan epitopes, but only Chklp regulates quorum sensing and oxidative stress adaptation. In addition, we have determined that the Slnlp and response regulator protein of C. albicans, Ssklp, regulate expression of a CHK1-promoter-lacZ reporter suggesting that the HOG1 MAP kinase pathway may interact with Chklp. These functional attributes and regulation of Chklp are new to studies of any two-component signal protein. Our overall objective is to focus upon Chklp in cell wall biosynthesis and other processes such as oxidant stress adaptation, as well as to identify the commonality of events regulated by Chklp and Hogtp. There are three specific aims in this proposal.
In specific aim 1, we will differentiate the primary lesion from secondary affects in the cell wall disassembly that are associated with the deletion of CHK1.
This aim will be achieved by using the tetracycline regulatable promoter. Also in aim 1, point mutations in CHK1 will be introduced to distinguish functions of several key domains. The second specific aim will utilize proteomics, microarrays, and tandem affinity chromatography [TAP] to identify proteins critical to the functions of Chklp (Calderone lab) as well as Hoglp (Pla lab).
Aim 2 includes important collaborations with the labs of J. Brown (TAP) and J. Kerwin (Proteomics).
Specific aim 3 will focus upon the functional response relationships of Chklp with Hoglp and include studies on nuclear translocation of Hoglp in the context of the chkl mutant. Epistasis experiments are designed to examine Chk1p-Hog1p interactions. The long-term objective of this research is to increase our understanding of the biological roles and contribution to disease of 2-component signal transduction in C. albicans, especially Chklp.
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